The present invention relates to compositions and methods for introducing genetic material into cells. More particularly, the present invention relates to compositions and methods for in vitro and in vivo gene transfer, which can be used to deliver protective and/or therapeutic agents including genetic material that encodes protein targets for immunization and therapeutic proteins.
The direct introduction of a normal, functional gene into a living animal has been studied as a means for replacing defective genetic information. DNA can be introduced directly into cells of a living animal using viral vectors, liposomes, lipid complexes, ligand/DNA conjugates, and microprojectile bombardment, among other methods. Various methods and compositions for mediating transfer of DNA to cells in vivo and/or in vitro are referred to in U.S. Pat. No. 5,593,972, issued Jan. 14, 1997; U.S. Pat. No. 5,580,859, issued Dec. 3, 1996; U.S. Pat. No. 5,589,466 issued Dec. 31, 1996; U.S. Pat. No. 5,676,954, issued Nov. 19, 1996; International Patent Publications Nos. WO90/11092, published Mar. 21, 1990; WO93/17706, published Mar. 10, 1993; WO93/23552, published May 21, 1993; and WO94/16737, published Jan. 26, 1994, and the priority applications cited therein.
Despite the knowledge extant in the art, there remains a need for improved methods of DNA transfer, as well as for improved methods and compositions for in vivo and in vitro nucleic acid transfer. There remains a need for improved methods of drug delivery.
In one aspect, the invention provides soluble, ionic complex comprising an aqueous mixture of a benzylammonium group-containing surfactant and a polynucleic acid sequence. In one embodiment, the complex is a vesicular-like or liposomal-like complex comprising an aqueous mixture of a benzylammonium group-containing surfactant of the formula described herein and a polynucleic acid sequence, with the sequence substantially packaged in the vesicular complex.
In another aspect, the invention provides a mixture of multiple ionic and/or vesicular complexes of uniform size, as above described. In one embodiment, the composition is formed by mixing an aqueous solution of a benzylammonium-containing surfactant, preferably benzalkonium chloride, with a polynucleic acid sequence.
In still another aspect, the invention provides a pharmaceutical composition comprising at least one, and preferably multiple ionic complexes or vesicular complexes described above and a suitable pharmaceutical carrier.
In yet another aspect, the invention provides a method of introducing a polynucleic acid sequence into a cell comprising the step of contacting said cell with the above described complexes or compositions containing them.
In another aspect, the invention provides a method of facilitating the uptake of a polynucleic acid sequence into a cell comprising contacting the cell with a soluble ionic complex described above, or with a polynucleic acid substantially packaged in a vesicular complex formed by an aqueous mixture of a benzylammonium-containing surfactant with the polynucleic acid sequence.
In a further aspect, the invention provides methods of inducing an immune response in a mammalian or vertebrate subject to a pathogenic antigen or disease, which methods include the step of administering to cells of said subject, an effective amount of a complex as described herein, wherein the polynucleic acid sequence encodes at least one epitope that is identical or substantially similar to an epitope of a antigen of said pathogen, or a sequence encoding a target protein, said protein comprising an epitope identical or substantially similar to an epitope of a protein associated with cells that characterize said disease. The epitope or protein-encoding sequence is under the control of regulatory sequences that direct expression of said protein in the cells of said subject.
In still another aspect, the invention provides a method of treating a mammalian or vertebrate subject for a disease comprising the step of administering to cells of said subject, an effective amount of a composition comprising a complex of this invention formed by an aqueous mixture of a benzylammonium-containing surfactant and a polynucleic acid sequence, wherein said polynucleic acid sequence comprises a sequence which encodes a protein that produces a therapeutic effect on the subject or a protein that compensates for a missing, non-functional or partially functioning native mammalian protein, the protein-encoding sequence under the control of regulatory sequences that direct expression of said protein in the cells of said subject.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.